DESCRIPTION: While bacterial infections present major threats to human health, antibacterial discovery has been difficult, particularly with the single-enzyme-based strategy. Two main reasons stand out: the occurrence of resistance mutations in the single enzyme target, and the insufficient chemical diversity of compound libraries used for screening inhibitors of the single enzyme target. A recent genome-wide analysis has ranked the enzyme TrmD as a leading antibacterial target, because it is essential for bacterial growth, broadly conserved across bacterial species, distinct from its human counterpart, and has a druggable site that drug-like molecules mimicking S- adenoscyl methionine (AdoMet) can bind to. TrmD is unlike targets of clinical antibiotics (the ribosome, DNA gyrase and topoisomerases, and cell-wall biosynthesis enzymes). Instead, TrmD is a tRNA enzyme that modifies G37 to m1G37 using AdoMet as the methyl donor. We hypothesize that TrmD is attractive for singe- enzyme-based drug discovery; because targeting TrmD would reduce bacterial efflux, allowing intracellular accumulation of multiple drugs for rapid cell killing before the occurrence of resistance. We also suggest that drugs targeting TrmD must explore novel chemical space and diversity. While pharmaceutical companies AstraZeneca (AZ) and GlaxoSmithKline (GSK) have made intense efforts to target TrmD as a member of growth-essential enzymes in bacteria, their anti-TrmD program has stalled, in part due to the use of radioactive 3H-AdoMet in a high-throughput screening (HTS) assay. We propose instead to develop and optimize a novel fluorescence assay that is more robust and cost-effective and is based on a principle different from that of the 3H assay. The development of this fluorescence assay will enable discovery of novel classes of inhibitors to probe how targeting TrmD can cause collateral damage on bacterial efflux in an innovative growth arrest mechanism distinct from the mechanisms of antibiotics in clinical use. Using E. coli TrmD (EcTrmD) as a model, preliminary work has validated the robustness and amenability of the fluorescence assay to the HTS format. Aim 1 will further improve parameters of the assay. Aim 2 will validate the assay for HTS-ready by collaboration with the NSRB/ICCB-Longwood (NSRB/ICCB-L) screening facility at Harvard. Following the validation, we will launch a large-scale screening campaign at Harvard to screen ~500,000 compounds from diverse chemical libraries. False positives will be removed in counter screens and the hit pool will be screened using secondary, tertiary, and phenotypic assays. Aim 3 will validate hits for the ability of targeting EcTrmD in the whole cell and will improve qualities of hits by chemical optimization based on our recently developed tRNA-bound crystal structure of TrmD in complex with sinefungin (a non-reactive analog of AdoMet). Hits with desired criteria will be tested for growth arrest and in vivo efficacy. The identified hits will serve as chemical probes to understand the growth-arrest mechanism of targeting TrmD and as leads for antibiotic discovery to address the global burden of bacterial infectious disease.